DNA Metabolism Flashcards
(115 cards)
1
Q
What did the Meselson Stahl Experiement prove?
A
The semiconservative model of DNA replication
- The first division ruled out conservative because all DNA was of intermediate density
- The second division ruled out dispersive model because DNA was either of intermediate or light density
2
Q
What is DNA polymerase?
A
Synthesize a complementary DNA strand in the 5’ to 3’ direction
It reads a DNA in the 3’ to 5’
DNA-dependent DNA polymerase
3
Q
What is the leading strand in a replication fork?
A
The strand synthesized in the same direction as fork movement in the 5’ to 3’ direction
4
Q
What is the Lagging strand in the replication fork?
A
The strand synthesized in the 5’ to 3’ in the opposite direction as fork movement
5
Q
What are some characteristics of the lagging strand?
A
Can’t be synthesized continuously
Synthesized as small fragments called Okazaki fragments which are later ligated
6
Q
What are Okazaki fragments?
A
Small fragments of newly synthesized DNA in the lagging strand of a replication fork
7
Q
DNA synthesis is semi-continuous, what does this mean?
A
Leading strand synthesis is continuous
Lagging strand synthesis is discountinuous
8
Q
Where are the only spots that replication can begin?
A
At the “origins of replication”
9
Q
From every origin of replication, how many replication forks are formed?
A
Two replication forks begin, migrating in opposite directions
10
Q
What doe DNA polymerase require in order to start DNA synthesis?
A
Require an existing 3’-OH to add nucleotides to provided by a primer
11
Q
When two replication forks open, where are the leading strands?
A
One strand will be the leading strand for one fork but the lagging strand for the other fork
(Leading strands are on opposite sides replicating away from each other)
12
34
E.g 1 and 4, 2 and 3 if 1,3 is a replication fork and vice versa
12
Q
What reaction causes DNA chain elongation?
A
Transesterification
An irreversible reaction due to how much energy was used. Hydrolysis contributes to the energétics
13
Q
What substrate adds the base to the elongating DNA chain?
A
Deoxynucleotidetriphosphates (dNTPS)
14
Q
What is the reaction mechanism on dNTP for DNA chain elongation? (Simple)
A
Nucleophilic attack by 3’ -OH of primer on first phosphate (alpha) of the incoming dNTP
The 2nd and 3rd phosphates (beta and gamma) are release as inorganic pyrophosphate (PPi)
15
Q
What is processivity?
A
The ability of an enzyme to catalyze multiple reaction without releasing its substrate
16
Q
What is an example of a processivity enzyme?
A
DNA polymerase
-This process is faster than distributive enzymes
17
Q
What are distributive enzymes?
A
Enzymes that disassociate from substrate and product after catalyzing their reaction
18
Q
DNA polymerase have varying processivity, Which are high and low?
A
DNA polymerase III has high processivity
DNA polymerase I has low processivity
19
Q
What are sliding clamps (associated with polymerase)
A
Highly processive DNA polymerase use sliding clamps to remain associated with their templates
Loading of sliding clamps require ATP hydrolysis
20
Q
How often do DNA polymerase make mistake?
A
Every 10k to 100k bp
21
Q
How are the mistakes of DNA polymerase removed?
A
Proofreading
(Separate activity from DNA polymerase)
A 3’-5’ exonuclease activity
22
Q
What are the 2 mechanisms to detect incorrect nucleotides?
A
- Mispairing results in weaker H-bonding and brings new strands to exonuclease site of polymerase
- Mispairing inhibits translocation of DNA through polymerase. The pause allows the new strand to enter the exonuclease site
(Shifts backwards a little bit)
23
Q
By how much does proofreading improve DNA synthesis?
A
Improves fidelity of replication by 100-1000 fold
24
Q
How many origins do bacteria have?
A
One replication origin: oriC
This site is highly conserved across bacteria species
25
What is the bacteria replication origin rich in?
High in GATC-methylated adenosines
2 regions with repeats
-9 by repeats, 12bp AT rich repeats
26
What initiates strand separation in bacteria at the AT-rich repeats?
DnaA (initiator protein)
27
Where does DnaA bind to?
The 9bp repeats
28
What is DnaC?
Helicase loading protein
Associates with DnaA and helps recruit DnaB
29
What is DnaB and what's it do?
Helicase enzyme
It further separates the strands and 2 replication forks begin? (DnaA initiates the separation)
30
When does initiation not occur at replication origins?
When the strands are hemimethylated origins
-it takes around 20 minutes to full methylate
31
When can DNA replication occur after the strands are open in bacteria?
When DNA becomes fully methylated
| Hemi looks like an eye with eyelashes and fully has strands on both side going up and down
32
What is DNA helicase?
Enzymes that separate (unwind) the strands of dsDNA
They bind one strand, translocate along it and displace the other strand
33
What does DNA helicase induce?
Induces positive supercoils
This is resolved by topoisomerase
34
What does DNA helicase require in order to work?
Requires energy to break the H-bonds
ATP hydrolysis: ATP -> ADP + Pi
35
Wha do single-stranded DNA binding proteins do?
1. Protect single-stranded DNA from nuclease attack
2. Prevent reannealing
3. Aid helicase by destabilizing the double helix
36
What does primase do? Bacteria
Generates 8-10bp of RNA to as a primer
Associates transiently with the helicase to form a primosome
Generates primers periodically on the lagging strand as the replication fork moves
37
What is the primary DNA polymerase in bacteria?
DNA polymerase III
38
What is the difference of okazaki fragments in bacteria vs eukaryotes?
Bacteria 1k-2k bases
Eukaryote are 100-200
39
What is the activity of DNA polymerase III?
Synthesize the leading and lagging strands
* 5'-3' DNA polymerase (DNA synthesis)
* 3'-5' exonuclease (proofreading)
40
What is the replisome? (Bacteria)
A complex made by the 2 DNA polymerase (for the leading and lagging strands)
41
What is the trombone model?
The lagging strand loops out as it is replicated so the polymerase can follow the replication fork
42
What periodically creates new RNA primers on the lagging strand? (Bacteria)
Primase in the primosome
43
After a primase adds a new RNA primer to the DNA template, what is loaded to the end of the new primer?
A new sliding clamp is loaded at the end of the new primer
44
What happens when DNA polymerase III on the lagging strand hits the preceding RNA primer? (Bacteria)
It releases the DNA strand
45
What is a nick?
An adjacent nucleotide not connected by a phosphodiester bond
46
What does DNA polymerase I do? (Bacteria)
Bind to the nick and removes the RNA
-It has a 5'-3' exonuclease activity for the nick
Synthesis DNA in the space (5' to 3')
Then proofreads (3'to5')
47
What does DNA ligase do? (Bacteria)
Closes the final nick between the DNA fragments
48
What does DNA ligase do? (Bacteria)
Catalyzes the formation of phosphodiester bond between:
5'-phosphate on DNA chain synthesized by DNA polymerase III
3'-OH on DNA chain synthesized by DNA polymerase I
49
What are the requirements for DNA replication? (Bacteria)
1. Template DNA
2. Primer
3. All four dNTPs
4. Proteins
(Initiator proteins, DNA polymerase, sliding clamp + loader, topoisomerase, helicase, primate, ss DNA binding proteins, nucleares, ligase)
50
In eukaryotes, when does replication occur?
Only during S phase
51
What controls transition between stages in eukaryotic cell cycle?
Checkpoints
| E.g. G1/S checkpoint controls entry into S phase
52
What initiates replication in eukaryotes?
Origin of replication protein complex (ORC)
Binds to origin of replication, (initiation)
This functionally replaces DnaA (bacterial)
53
What is the single-stranded DNA binding protein in eukaryotes?
RPA
54
What is helicase in eukaryotes?
Minichromosomes Maintenance complex (MCM)
-this functionally replaces DnaB
Licensing factor*?
55
What is Cdc6?
Recruits the helicase (MCM) (replaces DnaC)
This is the Matchmaker factor
Levels fluctuate though the cell cycle, controlled by cycling acting via cyclin-dependent kinases (CDKs)
Rapidly degraded and replication is inactive
56
What is DNA polymerase alpha?
A complex containing:
A. Primase activity
B. A DNA polymerase activity
Synthesize a short RNA primer (~10nt) then switches to DNA synthesis
No proofreading
57
What is DNA polymerase E?
An alternative processive polymerase
| Also involved in leading and lagging strand synthesis
58
What is DNA polymerase delta?
The primary, highly processive polymerase
Synthesizes the leading and lagging strand
Uses a sliding clamp called PCNA
And a clamp loader RFC
59
What is the sliding clamp and loader used by DNA polymerase delta?
Clamp: PCNA
Loader: RFC
60
What is the primary, highly-processive polymerase in eukaryotes?
DNA polymerase delta
61
What polymerase displace the RNA primer and continue synthesizing DNA, replacing the primer?
Delta and E
62
What is the RNA "flap" degraded by?
FEN1
63
What degrades RNA in RNA:DNA hybrids?
RNaseH
64
What does DNA ligase do in eukaryotes?
Closes the final nick between the DNA strands after the RNA primer has been removed
65
What does Reverse transcriptase do?
Read an RNA template 3'-5' and synthesizes a complementary DNA strand 5'-3'
66
What are reverse transcriptase carried by?
Retroviruses such as HIV and Hepatitis viruses
67
What are some characteristics of reverse transcriptase?
RNA-dependent DNA polymerase
No proofreading
68
What is cDNA (complementary DNA)?
A DNA copy of a mRNA
Generated using reverse transcriptase
69
What kind of sequence do cDNA's contain?
The entire coding sequence for a protein but no introns
-introns are removed during mRNA splicing
70
What is chromosome shortening associated with?
Aging
71
What is replication senescence?
After a certain number of cell division (hayflick limit) telomeres shorten to a critical length and stop dividing
72
What act as buffers for the end replication problem?
Telomeres TTAGGG repeats
-Telomeres shorten rather than important genetic information in the chromosome
73
What is the End replication problem?
Synthesis of the new stands can't extend completely to the 5' end
-there is no 3'-OH available for binding
74
What is a telomerase?
RNA-dependent DNA polymerase
A ribonucleoprotein
It is not active in adult somatic cells
75
When is telomerase active?
In germ cells, fetal cells, embryonic stem cells DNA cancer cells
76
What is a ribonucleoprotein?
A complex of protein and RNA
77
What does telomerase do?
Use RNA asa a template to extend the telomeres repeat DNA from the 3' end of chromosomes
- only one strand is synthesized
- the other strand is synthesized by normal DNA replication
78
What are the types of excision repair?
1. Mismatch repair
2. Nucleotide excision repair
3. Base exclusion repair
79
How does excision repair work ? (Steps)
Damage on 1 strand
A segment on the damaged strand is excised
A DNA polymerase fills in gap using the intact strand as a template
A Ligase seals the final nick
80
What doe slipped strand mispairing (or slippage) result in?
Insertions or deletions at tandem repeats
81
What does backward/forwards slippage results in?
Backward slippage causes insertion
Forward slippage causes deletion
82
How does bacterial mismatch detect which strand is the parent strand?
Based on the differential methylation done shortly after replication
83
What are the proteins in bacteria that detect mismatch?
Muts and MutL recognize mismatches or small insertion or deletion loops
They form a complex at the site of mutation
84
What does MutH bind to?
Hemimethylted site
85
What interactions with MutH after it binds to the hemimethylted site?
The MutS/MutL complex
MutH then cleaves the unmethylated strand (new)
86
After MutH removes the mismatch what occurs?
An exonuclease then digest the new strands from the nick just past the mismatch
DNA polymerase III fills in the gap and a ligase selas the final nick
87
What does mismatch repair raise the fidelity by?
Mismatch repair raises the fidelity of DNA synthesis to 1 error per billion bases
-1 error every 1.6 cell divisions
88
How does eukaryotic mismatch repair differentiate between the new and old strand?
By the presentes of single strand breaks in the new strand
- lagging strand: Okazaki fragments
- leading strand the 3' end?
89
What proteins recognize mismatch in eukaryotes?
MSH and MLH
90
What mutations are associated with Hereditary Nonpolyposis Colon Cancer (HNPCC)?
Mutations in MSH1 And MLH1
91
What does the Deamination of a 5'-methylcytosine produce?
Thymidine
-This results in a T-G mismatch
92
What happens when mismatch repairs occurs outside the window for discrimination?
A random base will be replaced
93
Most Species have a higher content of what pair of nucleotides according to Chargaff's rules?
AT content
94
What does Nucleotide excision repair do?
Repairs bulky lesions to a single strand
E.g.
- pyrimidine diners
- bulky groups
95
What are the two mechanisms of initiation for nucleotide excision repair?
1. Transcription couple NER
| 2. Global genomic NER
96
What does Transcription couple NER do?
Preferential repair of template strand
RNA polymerase stall at the site of damage
CSA and CSB bind damage*
97
What bind the damage for Transcription coupled NER?
CSA and CSB
98
What does Global genomic NER do?
Repair of either strand of XPC acts as a damage sensor
99
What do excision endonucleases do?
Cut the damaged strand on both sides of lesion
100
What do exinucleases do?
Make 2 cuts (usually nicks in only one strand)
The gap is filled in by a polymerase and the final nick is sealed by a ligase
101
What proteins remove damage segments in nucleotide excision repair?
XPB and XPD (they are helicases)
102
What is an autosomal recessive disease caused by the inability to repair pyrimidine dimers?
Xeroderma Pigmentosum (XP)
103
What is Xeroderma Pigmentosum?
An autosomal recessive disease cause by mutations in seven XPA-XPG genes
XPC mutations are MC
Unable to repair pyrimidine dimers
104
What are the clinical presentations for xeroderma Pigmentosum?
Photosensitivity
- severe burns
- skin blisters and lesion
- dry skin
- changes in pigmentation
2k-5k more risk for malignant melanoma
105
What is cockayne syndrome?
An autosomal recessive disease caused by mutations in CSA or CSB
Defect in transcription-coupled NER
1 in 200-300k
106
What are the clinical presentation of cockayne syndrome?
```
Photosensitivity
Failure to thrive, very short stature
Developmental abnormalities
No increased cancer risk*
Mean life expectancy= 3 years
```
107
What is base excision repair?
Repairs specific damage to bases
E.g. Methylation, deamination, oxidation
108
What does DNA glycosylases do in base excision repair?
Recognize the damaged base and cut glycosidic bond
-generate an AP site (apurinic or apyrimidinic)
109
What does DNA glycosylase do in Base excision repair?
Recognize uracil in the DNA from deamination of cytosine
A specific uracil DNA glycosylase associated with the replicó e to remove uracil incorporated during replication
110
What nicks the DNA in base excision repair? Removes the DN and fills the gap?
AP endonuclease nicks the DNA
An exonuclease removes a stretch of DNA from the damaged strand
A DNA polymerase fills in the gap and ligase seal the final nick
111
What are the causes of double stranded breaks in DNA?
Hydroxy radicals (ionizing radiation or oxidative phosphorylation)
DNA replication of damaged DNA
Chemotherapeutic drugs. E.g. Bleomycin
112
What are the 2 mechanisms for double strand break repair?
1. Non-homologous end joining (NHEJ)
- error prone
2. Homologous recombination
- error free
113
When both strand are broken, no intact strand can act as a template for re-synthesis of the damaged strand which may lead to?
More likely errors
Mutagenesis
114
How does Non-homologous end joining work?
Ku heterodimer binds to DNA ends
Recruits DNA-dependent protein kinase (DNA-PK)
Phosphorylation substrates bring the ends together
Ligation by DNA ligase IV
115
Defects in double stranded break repair pathways are associated with?
Several rare disorders featuring a predisposition to cancer
E.g
- ataxia telangiectasia (ATM gene)
- bloom syndrome (BLM gene)
- Nijmegen breakage syndrome (NBM)
- breast and ovarian cancer (BRCA1 and BRCA2)
